PROJECT SUMMARY Our laboratory has developed novel culture methods using engineered Notch ligands to increase the number of CD34+ precursors, including those able to provide rapid hematopoietic engraftment in patients undergoing cord blood (CB) transplantation. Clinical trials have determined the safety and effectiveness of this approach, but indicate the need to derive greater hematopoietic stem and progenitor cell (HSPC) numbers for a more potent, economically feasible therapy. Consequently, the focus of this grant is to elucidate new strategies to regulate Notch-induced signals to maximize HSPC growth versus differentiation. To this point, our CB CD34+ expansion protocol has focused on regulating Notch signal strength by controlling ligand availability, while largely neglecting the contribution of variation in cell-surface Notch paralog expression and susceptibility to activation. Recent observations compel us to investigate the use of paralog-specific activating antibodies to tightly regulate the level of Notch signal strength generated during the evolution of cell surface Notch expression that accompanies ex vivo culture (Aim 1). These studies also suggest that paralog-specific antibodies raised against the amino-terminus of the Notch extracellular domain induce Notch activation in stem cell populations insusceptible to Notch ligand-induced activation by overcoming the inhibition of Notch activation due to endogenous Notch ligand expression (cis-inhibition). We will confirm that differential agonist potency in promoting Notch activation stems from ligand-mediated cis- inhibition and determine whether use of Notch antibody agonists to overcome this suppression enhances the generation of engraftable HSPC (Aim 2). These studies will provide insight into the cell-autonomous mechanisms regulating Notch receptor function, an outcome of conceptual and practical interest to those focused on manipulating cell-fate decisions in Notch-dependent stem cell types. Lastly, we will combine our findings maximizing Notch-induced HSPC self- renewal while preventing differentiation with the activation of signaling pathways promoting HSPC survival, allowing further expansion of HSPC (Aim 3). Together, these aims advance us toward our long-term goal of developing novel strategies for stem cell engineering, with a focus on achieving a substantial increase in HSPC expansion for therapeutic purposes.